Synthesis and hLDH Inhibitory Activity of Analogues to Natural Products with 2,8-Dioxabicyclo[3.3.1]nonane Scaffold

Human lactate dehydrogenase (hLDH) is a tetrameric enzyme present in almost all tissues. Among its five different isoforms, hLDHA and hLDHB are the predominant ones. In the last few years, hLDHA has emerged as a therapeutic target for the treatment of several kinds of disorders, including cancer and primary hyperoxaluria. hLDHA inhibition has been clinically validated as a safe therapeutic method and clinical trials using biotechnological approaches are currently being evaluated. Despite the well-known advantages of pharmacological treatments based on small-molecule drugs, few compounds are currently in preclinical stage. We have recently reported the detection of some 2,8-dioxabicyclo[3.3.1]nonane core derivatives as new hLDHA inhibitors. Here, we extended our work synthesizing a large number of derivatives (42–70) by reaction between flavylium salts (27–35) and several nucleophiles (36–41). Nine 2,8-dioxabicyclo[3.3.1]nonane derivatives showed IC50 values lower than 10 µM against hLDHA and better activity than our previously reported compound 2. In order to know the selectivity of the synthesized compounds against hLDHA, their hLDHB inhibitory activities were also measured. In particular, compounds 58, 62a, 65b, and 68a have shown the lowest IC50 values against hLDHA (3.6–12.0 µM) and the highest selectivity rate (>25). Structure–activity relationships have been deduced. Kinetic studies using a Lineweaver–Burk double-reciprocal plot have indicated that both enantiomers of 68a and 68b behave as noncompetitive inhibitors on hLDHA enzyme.


Introduction
Lactate dehydrogenase (LDH; EC 1.1.1.27) is an enzyme widely distributed in nature that belongs to the 2-hydroxyacid oxidoreductases family. It is a tetrameric molecule (140 kDa) mainly formed by two different kinds of subunits of 35 kDa each: the M-type (muscle) subunit encoded by Ldha gene and the H-type (heart) encoded by Ldhb gene. The combination of both subunits provides up to five different LDH isoforms: H 4 (also named LDH-1 or LDHB), H 3 M (LDH-2), H 2 M 2 (LDH-3), HM 3 (LDH-4), and M 4 (LDH-5 or LDHA). All of them show different kinetic behaviors and tissue distributions. The homotetramers LDHA and LDHB are the major isozymes that are present in human cells and are mainly located in (i) skeletal muscle and liver, and (ii) in heart and brain tissues, respectively [1,2].
This enzyme plays an important role in several metabolic pathways where it regulates the homeostasis of pyruvate/lactate, hydroxypyruvate/glycerate, and oxalate/glyoxylate, using NADH as cofactor. One of the main and most studied roles of LDH enzymes is the regulation of the energy supply mechanism in cancer and normal cells (Warburg effect) [3].
Non-cancer cells obtain their energy demand by the complete metabolism of glucose, which is performed in two subsequent steps: (i) conversion of glucose into pyruvate (glycolysis), and (ii) degradation of the generated pyruvate to CO 2 (tricarboxylic acid cycle (TCA)). The complete catabolism of glucose allows a sustainable energy production system due to the generation of ATP molecules and the regeneration of NAD + cofactor during the oxidative phosphorylation (OXPHOS).
LDH and pyruvate dehydrogenase kinase (PDHK) enzymes are in charge of regulating the flux of pyruvate into TCA cycle [4]. The LDH function becomes very important when oxygen supply is reduced and/or cells increase their glucose demand and, therefore, the fermentation pathway is activated. In this case, pyruvate is reduced into lactate, regenerating NAD + and producing ATP molecules but in a less efficient way. This last reaction is catalyzed by LDH enzymes and allows the maintenance of the equilibrium when glucose uptake is increased.
On the opposite hand, cancer cells need precursors to build up macromolecules in a quicker way. In that sense, and trying to fulfill this requirement, they increase their glucose uptake and adjust its metabolism in favor of a quicker but less efficient process of obtaining energy [5], in which lactic acid fermentation and LDH enzymes play the main role even in the presence of oxygen (aerobic glycolysis). As a result, high levels of glycolytic metabolites are formed, which can be used for the quick development of cancer cells [6]. This change in the metabolic glucose pathway causes the need for overexpressed LDH enzymes in cancer tissues, turning these enzymes, and particularly LDHA isozyme, into a possible therapeutic target for the development of new anticancer therapies [7].
Moreover, apart from its relevance in cancer, human LDHA isozyme (hLDHA) has recently attracted great interest among scientists, due to its proven important role in molecular mechanism of different kind of disorders [8], such as vascular diseases [9,10], epilepsy [11,12], tuberculosis [13], pulmonary fibrosis [14], arthritis [15] and other inflammatory diseases [16,17], and in primary hyperoxalurias (PHs) [18]. Recent advances in the understanding of the molecular mechanisms of several diseases have led to the development of novel therapeutic approaches, such as enzyme deletion using in vivo CRISPR/Cas9 [19][20][21] or mRNA silencing using siRNA [22][23][24][25][26]. However, pharmacological treatments based on small-molecule drugs could present some advantages compared to the use of biotechnological approaches. The cost of production is generally lower, and they can be administered orally and present better ADME (absorption, distribution, metabolism, and excretion) properties. In any case, a combined treatment with both types of pharmacological agents could have synergistic effect and benefits, such as lowering the required doses or the frequency of administration. Actually, approaches based on attenuated expression of LDHA or inhibition of LDHA activity by small-molecule drugs appear as emerging strategies for the treatment of these diseases [27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43]. Pharmaceutical companies and researchers in academia are investing significant efforts to identify natural or synthetic hLDHA inhibitors with a huge structural variability. Although some of them have shown EC 50 values in cancer cell line at nanomolar range [33], limitations related to ADME and pharmacokinetic properties have reduced the number of compounds evaluated in vivo and, to the best of our knowledge, none of them have progressed towards clinical trial [27,28].
Our research group is also interested in the development of hLDHA inhibitors as a pharmacological option for the treatment of PHs, a rare life-threatening genetic disease [34,35]. We recently reported the detection of (±)-2,8-dioxabicyclo [3.3.1]nonane derivatives, analogues to A-type proanthocyanidin natural products, as a new family of hLDHA inhibitors [35]. That research was inspired by the results obtained by Li et al., who performed LDHA inhibition assays on procyanidin-enriched fractions from Spatholobus suberectus extract [44], and by our experience in the synthesis of compounds with this bicyclic scaffold [45][46][47]. Thus, we have reported the synthesis of twenty (±)-2,8dioxabicyclo [3.3.1]nonane derivatives (1-20) (Figure 1), their hLDHA and hLDHB inhibitory activities, and the ability of three of them (2, 3 and 9) to reduce the quantity of oxalate generated by hyperoxaluric mouse hepatocytes (PH1, PH2, and PH3) in vitro [35]. These results encouraged us to continue exploring this family of bicyclic compou and to synthesize and evaluate a more complete collection of 2,8-dioxabicyclo[3.3.1]n ane derivatives in order to establish structure−activity relationships that allow to un stand the main features that bicyclic core should fulfil to ensure high potency and s tivity towards hLDHA.

Chemical Synthesis
Our research group previously synthesized (±)-2,8-dioxabicyclo[3.3.1]nonane de atives by reaction of flavylium salts with phenolic nucleophiles, such as phloroglucin resorcinol [45,47]. The fact that these compounds have emerged as a new family of hLD selective inhibitors [35], encouraged us to systematically design a series of 2,8-dioxab clo[3.3.1]nonane compounds in order to gain insight into the structural requirements are responsible for hLDHA activity and hLDHA/hLDHB selectivity. In that sense, 44 b clic derivatives (1-6; 42-70) (35 are new compounds) have been prepared by reaction tween 9 different flavylium salts (27-35) (3 are new compounds), with modifications a and B-rings, and phloroglucinol (36) and other non-phenolic π-nucleophiles (37 (Scheme 1). Nucleophiles 36-38 are commercially available, but pyrone-type compou 39-41 have been prepared as racemic mixtures according to a previously reported m odology by reaction of the proper benzaldehyde derivative and ethyl acetoacetate [48 The yields obtained and the spectroscopic data of these pyrone derivatives are consis with the reported ones (39 (88%) [48], 40 (80%) [48], and 41 (74%) [48,50]). Flavylium salts were synthesized by an aldol condensation under acidic condit between salicylic aldehyde (21) or derivatives (22 and 23) and acetophenone derivat (24)(25)(26) according to procedures previously used by us [35,45,51]. Three different sub uents have been selected at position 6 of the A-ring (R1 at Scheme 1) with different tronic effects on the bicyclic core. Strong and weak electron withdrawing groups (EW NO2 and Cl, respectively) along with the absence of a substituent in A-ring have b employed. For the B-ring, OH and OMe groups have been selected at position 3′ and/ (R2 and R3 at Scheme 1), taking into account that hydroxyl groups always appear in p phenolic scaffold-based LDHA inhibitors (LDHAi's) [31]. All flavylium salts were tained with moderate to quantitative yields (63-99%; Table 1). It is worth noting tha three new flavylium salts with two OMe groups in B-ring (30)(31)(32) were obtained with highest yields (96-99%). These results encouraged us to continue exploring this family of bicyclic compounds and to synthesize and evaluate a more complete collection of 2,8-dioxabicyclo[3.3.1]nonane derivatives in order to establish structure−activity relationships that allow to understand the main features that bicyclic core should fulfil to ensure high potency and selectivity towards hLDHA.
Although inhibition potency (IC 50 against hLDHA) is an important parameter that characterize an inhibitor, its selectivity through the target enzyme governs its applicability and it is a feature that should be also taken into account. In order to know the selectivity of the synthesized compounds against hLDHA, their hLDHB inhibitory activities were also measured. In Figure 4, the inverse of IC 50 for each compound against both enzymes is represented for the sake of clarity. All of them showed higher inhibitory activity (higher 1/IC 50 values) against hLDHA than against hLDHB, except for compounds 1, 4, 46, 51, and 54. Although inhibition potency (IC50 against hLDHA) is an important parameter that characterize an inhibitor, its selectivity through the target enzyme governs its applicability and it is a feature that should be also taken into account. In order to know the selectivity of the synthesized compounds against hLDHA, their hLDHB inhibitory activities were also measured. In Figure 4, the inverse of IC50 for each compound against both enzymes is represented for the sake of clarity. All of them showed higher inhibitory activity (higher 1/IC50 values) against hLDHA than against hLDHB, except for compounds 1, 4, 46, 51, and 54.      (50), two with 2-hydroxy-1,4-naphthoquinone (58 and 59), and eight with a pyrone-type moieties (62a, 63a, 65a, 65b,  67a, 68a, 68b, and 70b). In particular, compounds 58, 62a, 65b, and 68a have shown the lowest IC 50 values against hLDHA (3.6-12.0 µM) and the highest selectivity rate (>25), which allow us to select them as hits for future structural optimization. These data seem to demonstrate that the use of pyrone derivatives as nucleophilic moiety is an important structural feature for ensuring high potency and selectivity against hLDHA. Regarding the behavior of both diastereomers of compounds 62-70, all of them showed better inhibition activity against hLDHA than against hLDHB (Figure 4), and in terms of selectivity ( Figure 5), a slightly higher selectivity is observed for the "cis" than for the "trans" isomers, with the exception of compounds 65 and 70 ( Figure 5). This means that a clear influence of the relative arrangement of rings B and E of compounds 62-70 cannot be established yet with the current data.
According to these inhibitory activity and selectivity results, a preliminary structureactivity analysis can be made. Figure 6 shows the number of compounds (in percentage) with a specific structural moiety (substituents at A-and B-rings and nucleophilic moiety) that meet the requirement of a selected inhibition activity (blue line, hLDHA inhibition activity lower than 30 µM; yellow line, hLDHB inhibition activity higher than 30 µM). It is deduced from the graphic that the presence of EWGs (NO2 and Cl) at A-ring is an important feature to ensure higher inhibition activity against hLDHA. Percentagewise, the number of compounds with a nitro or chloro substituent at A-ring and IC50 values against hLDHA lower than 30 µM is much higher than those without a substituent at A-ring. In addition, the number of compounds with chloro at A-ring and hLDHB inhibition activity higher than 30 µM is also higher than those with a nitro substituent. This means that derivatives with Cl at A-ring show better selectivity rate. Regarding the behavior of both diastereomers of compounds 62-70, all of them showed better inhibition activity against hLDHA than against hLDHB (Figure 4), and in terms of selectivity ( Figure 5), a slightly higher selectivity is observed for the "cis" than for the "trans" isomers, with the exception of compounds 65 and 70 ( Figure 5). This means that a clear influence of the relative arrangement of rings B and E of compounds 62-70 cannot be established yet with the current data.
According to these inhibitory activity and selectivity results, a preliminary structureactivity analysis can be made. Figure 6 shows the number of compounds (in percentage) with a specific structural moiety (substituents at A-and B-rings and nucleophilic moiety) that meet the requirement of a selected inhibition activity (blue line, hLDHA inhibition activity lower than 30 µM; yellow line, hLDHB inhibition activity higher than 30 µM). It is deduced from the graphic that the presence of EWGs (NO 2 and Cl) at A-ring is an important feature to ensure higher inhibition activity against hLDHA. Percentagewise, the number of compounds with a nitro or chloro substituent at A-ring and IC 50 values against hLDHA lower than 30 µM is much higher than those without a substituent at A-ring. In addition, the number of compounds with chloro at A-ring and hLDHB inhibition activity higher than 30 µM is also higher than those with a nitro substituent. This means that derivatives with Cl at A-ring show better selectivity rate. Regarding B-ring substituents, the percentage of compounds with two oxyg groups (R2=R3=OH or OMe) and IC50 values against hLDHA lower than 30 µM is than those with just one group (R2=OH; R3=H). Within this subgroup of deoxyg compounds, the percentage of cathecol derivatives (R2=R3=OH) with hLDHB inh activity higher than 30 µM is lower, pointing out a decrease in the selectivity wh hydroxyl groups are present.
Finally, the influence of the nucleophilic moiety on the inhibitory activity a hLDHA and on the selectivity rate is remarkable ( Figure 6). Percentagewise, the h number of compounds with low IC50 values against hLDHA corresponds to those w hydroxycoumarin (37), 4-hydroxy-6-phenyl-5,6-dihydro-2H-pyran-2-one (39), o chlorophenyl)-4-hydroxy-5,6-dihydro-2H-pyran-2-one (40) moieties. In particul substitution pattern presented in nucleophile 39 favors a greater number of comp with IC50 values against hLDHB higher than 30 µM, thereby increasing its selecti happen for compounds 62, 65, or 68.  Regarding B-ring substituents, the percentage of compounds with two oxygenated groups (R 2 =R 3 =OH or OMe) and IC 50 values against hLDHA lower than 30 µM is higher than those with just one group (R 2 =OH; R 3 =H). Within this subgroup of deoxygenated compounds, the percentage of cathecol derivatives (R 2 =R 3 =OH) with hLDHB inhibition activity higher than 30 µM is lower, pointing out a decrease in the selectivity when free hydroxyl groups are present.

Resolution of Racemates and Inhibitory
Finally, the influence of the nucleophilic moiety on the inhibitory activity against hLDHA and on the selectivity rate is remarkable ( Figure 6). Percentagewise, the highest number of compounds with low IC 50 values against hLDHA corresponds to those with 4-hydroxycoumarin (37), 4-hydroxy-6-phenyl-5,6-dihydro-2H-pyran-2-one (39), or 6-(4chlorophenyl)-4-hydroxy-5,6-dihydro-2H-pyran-2-one (40) moieties. In particular, the substitution pattern presented in nucleophile 39 favors a greater number of compounds with IC 50 values against hLDHB higher than 30 µM, thereby increasing its selectivity as happen for compounds 62, 65, or 68. Some of the most active and selective racemic compounds synthesized (66a, 68a, and 68b) were purified by chiral HPLC, using a Chiralpak IC column and hexane and dicholomethane (30:70, v/v) as mobile phase. The proper purity of each enantiomer has been also checked by HPLC (chromatograms of each pure enantiomer are included in the Supplementary Material Figures S127-S132), using a Chiralpak IC analytical column and by the measurement of their optical rotation. The inhibition activity of pure enantiomers has been determined against both enzymes, hLDHA and hLDHB ( Table 3). The dose response curves against hLDHA and hLDHB of each pure enantiomer are included in the Supplementary Material (Figures S199-S206). According to the results obtained, it seems that the spatial influence of the substituents in the selected 2,8-dioxabicyclo[3.3.1]nonane derivatives for the enzymatic inhibition assays is not very high due to the similar values of IC 50 obtained for each pair of enantiomers. Moreover, the inhibition activity of each single enantiomer was also very similar to the one obtained by its corresponding racemic mixture. For the hLDHA inhibition test, it seems that a better behavior is observed for dextrorotarory enantiomers, especially for compounds 66a and 68a (1.5 times more potent). For the hLDHB inhibition test, only compound 66a was active and both enantiomers showed also similar potency.

Resolution of Racemates and Inhibitory
It seems that the relative "cis-trans" arrangement of B and E ring in pyrone-type bicycle derivatives (62a-70a vs. 62b-70b) plays a more important role in the enzymatic inhibition assays performed than the different absolute configurations observed for pure enantiomers.

Mechanism of hLDHA and hLDHB Inhibition
In order to explore the mechanism of inhibition of some of the most active compounds on hLDHA, both enantiomers of the racemate 68a and 68b have been selected. Assays measuring the enzymatic activity of both compounds in the presence of four different inhibitor concentrations and without an inhibitor and eight different substrate (pyruvate) concentrations have been measured by a similar kinetic spectrofluorimetric procedure to that described to calculate IC 50 values against hLDHA (Section 3.4). The initial velocity (V 0 ) was determined as the maximum slope per minute calculated in the linear interval of the graph representing the progression of the enzymatic reaction at each inhibitor and substrate concentration. The progression of the enzymatic reaction was monitored by the decrease in fluorescence, due to NADH conversion into NAD + , registered at λ ex 340 nm and λ em 460 nm every 60 s during 10 min. Nonlinear fits of V 0 versus substrate concentration (Michaelis-Menten type) were performed for each inhibitor concentration and without inhibitor to determine V max and K M at each curve (values of V max and K M are included in the Supplementary Material Tables S1-S4). A decrease in apparent V max and no effect on the apparent K M were observed (with increasing concentrations of both enantiomers of racemate 68a and 68b), which is indicative of a noncompetitive inhibition. In addition, their corresponding Lineweaver-Burk double-reciprocal graphs showed intersection on the x axis, which is also favorable to this type of inhibition (graphs included in the Supplementary Material Figures S207-S210). Finally, a noncompetitive inhibition fit of V 0 versus substrate concentration allowed us to obtain the K i values, 4.1 and 16.1 µM for dextrorotatory 68a and levorotatory 68a, respectively, and 35.9 and 76.9 µM for dextrorotatory 68b and levorotatory 68b, respectively. Noncompetitive inhibition is not affected by the substrate concentration and this mechanism could suggest an allosteric binding to the enzyme, which has already been suggested by others [59].

General Experimental Methods
All solvents and reagents used in the synthesis or biological evaluations were purchased and were used as received, except MeOH, which was previously dried, using standard methodologies [60].
All reactions were conducted under inert conditions at room temperature or at 50 • C. Reactions were monitored by analytical thin-layer chromatography (TLC) on silica gel 60 F 254 precoated aluminum sheets (0.25 mm, Merck Chemicals, Darmsdadt, Germany) and spots were detected using UV light (λ = 254 nm). Purification steps of synthesized racemic compounds were carried out by column chromatography (CC) using Sephadex LH-20 or Silica gel 60 (particle size 0.040-0.063 mm) (Merck Chemicals, Darmsdadt, Germany). Purity grade of synthesized racemic compounds were performed on a Waters 1 H NMR and 13 C NMR spectra were measured on a Bruker Avance 400 spectrometer (Bruker Daltonik GmbH, Bremen, Germany) operating at 400 and 100 MHz for 1 H and 13 C, respectively. Deuterated methanol (CD 3 OD), deuterated chloroform (CDCl 3 ), or deuterated dimethylsulfoxide ((CD 3 ) 2 SO) was used as solvents. For flavylium salts a drop of TFA-d (trifluoroacetic acid deuterated) was added to obtain acidic conditions. Chemical shifts (in ppm) were referenced to solvent peaks as internal reference. The coupling constants (J) are expressed in hertz (Hz). The coupling system is described by using the following abbreviations: d, doublet; t, triplet; m, multiplet; br s, broad singlet; br d, broad doublet; dd, doublet of doublets; ddd, doublet of doublet of doublets; td, triplet of doublets; dq, doublet of quartets. The total assignment of 1 H and 13 C signals was made using 2D NMR spectra such as COSY, HSQC, and HMBC. High-resolution mass spectra (HRMS) were conducted on an Agilent 6520B Quadrupole time-of-flight (QTOF) mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with an electrospray ionization (ESI) interface operating in positive or negative mode.

Human Lactate Dehydrogenase A Enzymatic Activity Assay
The ability of the synthesized compounds to inhibit the hLDHA enzyme was measured using recombinant human LDHA (95%, specific activity >300 units/mg and concentration of 0.5 mg/mL, Abcam, Cambridge, United Kingdom) with sodium pyruvate (96%, Merck) as substrate and β-NADH (≥97%, Merck) as cofactor in a potassium phosphate buffer (100 mM, pH 7.3). The enzymatic assay was conducted on 96-well microplates and the decrease in the β-NADH fluorescence (λ excitation = 340 nm; λ emission = 460 nm) was detected in a TECAN Infinite 200 Pro M Plex fluorescent plate reader (Tecan Instrument, Inc.) at 28 • C. The activity was determined according to our previously described protocol [34,35]. The final volume in each well was set to 200 µL using 100 mM potassium phosphate buffer, 0.041 units/mL LDHA, 155 µM β-NADH, 1 mM pyruvate (saturated conditions), and DMSO solutions (5%, v/v) of pure compounds at concentrations in the range of 1-600 µM. The addition of pyruvate allowed the beginning of the reaction and fluorescence was registered every 60 s during 10 min. A lineal time interval was selected to calculate the slope at every single concentration. The establishment of the 0% and 100% enzymatic activity was performed by controls and by the use of the inhibitor 3-[[3[(cyclopropylamino)sulfonyl]-7-(2,4-dimethoxy-5-pyrimidinyl)-4-quinolinyl]amino]-5-(3,5-difluorophenoxy)benzoic acid (GSK 2,837,808 A, Tocris, MN, USA) at 1 µM [61]. The slope obtained at each concentration was compared to the one obtained for the 100% enzymatic activity control to determine the corresponding enzymatic activity. A nonlinear regression analysis in GraphPad Prism version 9.00 for Windows (GraphPad Software, La Jolla, CA, USA) was used for dose response curve, fitting of logarithm of inhibitor concentration vs. normalized enzymatic activity, to calculate IC 50 values (Supplementary Material section). All measurements were made in triplicate and data were expressed as the mean ± SD.

Human Lactate Dehydrogenase B Enzymatic Activity Assay
The ability of the synthesized compounds to inhibit the hLDHB enzyme was measured using recombinant human LDHB (95%, specific activity >300 units/mg and concentration of 1.0 mg/mL, Abcam, Cambridge, UK) following the same fluorimetric protocol described in Section 3.4.

Method for Determining the Mechanism of Inhibition on hLDHA
For each compound evaluated, three replicates of four different concentrations of the compounds in the presence of eight substrate concentrations were included in the kinetic fluorometric assay. The preparation of 96-well microplates and the reaction mixture in each well is the same as that described in Section 3.4. After the addition of the substrate, the maximum slope per minute, determined in a linear interval for each well, was used to establish the initial velocity (V 0 ) corresponding to each inhibitor and substrate concentration. Therefore, values of V 0 were obtained as a mean of three replicates. Nonlinear regression fits of V 0 versus substrate concentration have been performed in GraphPad Prism 9.00 for Windows (GraphPad Software, La Jolla, California, USA) to calculate V max , K M and K i . Linear Lineweaver-Burk double-reciprocal plots were created using Microsoft Excel 2019 (Microsoft Office Professional Plus 2019) to propose a mechanism of inhibition on hLDHA.
Most of the synthesized (±)-2,8-dioxabicyclo[3.3.1]nonane compounds showed higher inhibitory activity against hLDHA than against hLDHB. Nine of the new compounds (43, 44, 47, 58, 60, 62a, 65b, 66a, and 66b) and the reference compound 2, previously synthesized, showed IC 50 values against hLDHA lower than 10 µM. On the other hand, all of the synthesized compounds showed IC 50 values against hLDHB higher than 10 µM. In fact, only three of the new compounds (46, 51 and 54) presented a slightly higher inhibitory activity against hLDHB than against hLDHA. Quiral HPLC was used to separate enantiomers of a selection of the most active and selective compounds synthesized (66a, 68a, and 68b). The inhibition assays performed with these pure enantiomers revealed that the influence of the absolute spatial configurations in the inhibition behaviors of the selected 2,8-dioxabicyclo[3.3.1]nonane derivatives is not very high, although it seems that it is always favorable to dextrorotatory enantiomers on hLDHA. Moreover, the study of the inhibition kinetics of enantiomers of 68a and 68b seems to indicate a noncompetitive inhibition behavior for all cases and a slight preference for the dextrorotatory enantiomer (2-4 times more potent) during the inhibition kinetic process on hLDHA enzyme.
All these studies seem to indicate that 2,8-dioxabicyclo[3.3.1]nonane derivatives are promising inhibitors in terms of potency and selectivity against hLDHA enzyme. However, some additional tests have to be performed in order to reduce possible unspecific inhibition due to protein aggregation induced by the tested compounds (using a BSA/Triton enriched medium).
A structure-activity analysis has been conducted taking into account potency and selectivity against hLDHA and hLDHB enzymes, concluding that the presence of electronwithdrawing groups (NO 2 and Cl) at the A-ring, two methoxy groups at the B-ring, and preferably a pyrone moiety in the final compounds seems to be important features that 2,8-dioxabicyclo[3.3.1]nonane derivatives should fulfil in order to ensure achieving hLDHA inhibitors with high activity and selectivity (62a, 65b and 68a).